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(Hypertension. 1999;33:183-189.)
© 1999 American Heart Association, Inc.

Scientific Contributions

TNF-–Induced Migration of Vascular Smooth Muscle Cells Is MAPK Dependent

Stephan Goetze; Xiao-Ping Xi; Yasuko Kawano; Hiroaki Kawano; Eckart Fleck; Willa A. Hsueh; Ronald E. Law

From the Division of Endocrinology, Diabetes and Hypertension, University of California, Los Angeles, School of Medicine (S.G., Y.K., H.K., W.A.H., R.L.); and the Department of Medicine/Cardiology, Virchow Klinikum, Humboldt University Berlin and German Heart Institute, Berlin, Germany (S.G., E.F.).

Correspondence to Ronald E. Law, PhD, UCLA School of Medicine, Division of Endocrinology, Diabetes and Hypertension, Warren Hall, Second Floor, Suite 24-130, 900 Veteran Ave, Box 957073, Los Angeles, CA 90095. E-mail rlaw{at}med1.medsch.ucla.edu

	Abstract

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Abstract—Migration of vascular smooth muscle cells (VSMC) is a key event in neointimal formation and atherosclerosis that may be linked to the accumulation of inflammatory cells and release of chemotactic cytokines. Tumor necrosis factor- (TNF-) induces chemotaxis of inflammatory cells and fibroblasts, but little is known about chemotactic signaling by TNF- in VSMC. The aim of this study was to investigate the role of TNF- in VSMC migration and to elucidate the chemotactic signaling pathways mediating this action. TNF- (50 to 400 U/mL) induced migration of cultured rat aortic VSMC in a dose-dependent manner. Because activation of the extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (MAPK) is known to be required in platelet-derived growth factor–directed and angiotensin II–directed migration of these cells, we used the MAPK-inhibitor PD98059 to determine if chemotactic signaling by TNF- involves the MAPK pathway as well. We found that TNF-–directed migration was substantially inhibited by PD98059. TNF- (100 U/mL) transiently activated MAPK with a maximal induction 10 minutes after stimulation that returned to baseline levels by 2 hours after treatment. Only a single peak of increased MAPK activity was seen. PD98059 also blocked TNF-–stimulated MAPK activation in a concentration-dependent manner, which is consistent with its inhibition of TNF-–directed migration. To identify which TNF- receptor is involved in TNF-–induced MAPK activation, antibodies against the p55 TNF- receptor-1 (TNF-R1) and the p75 TNF- receptor-2 (TNF-R2) were used. VSMC express both receptors, but TNF-–induced MAPK activation was inhibited only by the TNF-R1 antibody. The TNF-R2 antibody had no effect. Thiazolidinediones are known to inhibit TNF- signaling in adipose tissue and attenuate platelet-derived growth factor–directed and angiotensin II–directed migration in VSMC. We therefore investigated the effects of the thiazolidinediones troglitazone (TRO) and rosiglitazone (RSG) on TNF-–induced migration. Both TRO and RSG inhibited migration, but neither attenuated TNF-–induced MAPK activation, indicating that their antimigration activity was exerted downstream of MAPK. These experiments provide the first evidence that early activation of MAPK is a crucial event in TNF-–mediated signal transduction leading to VSMC migration. Moreover, inhibition of TNF-–directed migration by the insulin sensitizers TRO and RSG underscores their potential as vasculoprotective agents.

Key Words: signal transduction • muscle, smooth • atherosclerosis • MAPK • cytokine • migration

	Introduction

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Vascular smooth muscle cell (VSMC) migration is a critical event in lesion development and progression in restenosis and atherosclerosis.¹ Migration of VSMC is a complex response that occurs during several pathological processes that involve accumulation of inflammatory cells and the release of chemotactic cytokines.² Cytokines such as tumor necrosis factor- (TNF-) are pluripotent mediators of inflammation³ and have been implicated in the chemotactic response of inflammatory cells⁴ and fibroblasts.⁵ Little is known, however, about the chemoattractant effects of TNF- in the vasculature. In addition to its role as an immune modulator, TNF- may also play an important role in atherogenesis and restenosis.⁶ TNF- has been found to be expressed in VSMC after balloon injury⁷ and in restenotic lesions⁸ but not in the normal vasculature. Moreover, the presence of TNF- has been demonstrated in intimal VSMC⁹ and in plaques of atherosclerotic arteries¹⁰ as well as in models of transplantation-associated atherosclerosis.¹¹ In an animal model of coronary-graft atherosclerosis, blockade of TNF- with a soluble TNF- receptor¹² inhibited acute coronary neointimal formation. Thus, cells of the arterial wall can both produce and respond to this cytokine in vivo. Thus TNF- is a highly important cytokine in the injured vasculature, where it may function to regulate the expression of growth factors (platelet-derived growth factor [PDGF], vascular endothelial cell growth factor [VEGF], fibroblast growth factor [FGF]),^{13 14} adhesion molecules,¹⁵ cytokines,¹⁶ and extracellular matrix degrading metalloproteinases¹⁷ as well as directly affect VSMC growth and migration. To better define the role of TNF- in the injured vasculature, we investigated its effect on VSMC migration and identified the signaling pathways mediating this process.

	Methods

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Materials
Dulbecco's modified Eagle's medium (DMEM), glutamine, antibiotics, HEPES, DMSO, and monoclonal antibody against smooth muscle -actin were obtained from Sigma (St Louis, Mo); rat recombinant TNF- was from R&D systems (Minneapolis, Minn); and Hybond enhanced chemiluminescence nitrocellulose membrane, horseradish peroxidase–linked anti-rabbit antibody, and enhanced chemiluminescence Western blotting detection reagents were from Amersham Life Sciences (Arlington Heights, Ill). Fetal bovine serum (FBS) was purchased from Irvine Scientific (Santa Ana, Calif). Culture plastic ware and cell culture inserts (8 µm, 24 well-format; Falcon) and transwell chambers were obtained from Costar. Cell fixation and staining was performed with the Quik-Diff stain set from DADE. Sprague-Dawley rats were from Charles River, Mass. The MAPK-ERK Kinase (MEK) inhibitor PD98059 and phosphospecific and total-extracellular signal-regulated kinase (ERK)1/ERK2 mitogen-activated protein kinase (MAPK) rabbit antibodies were purchased from New England BioLabs (Beverly, Mass). Troglitazone (TRO) was kindly provided by Parke Davis; rosiglitazone (RSG, formerly BRL 49653) was a generous gift from Smith Kline Beecham. Antibodies against TNF- receptor-1 and receptor-2 were obtained from Santa Cruz Biotechnologies (Santa Cruz, Calif). The bromodeoxyuridine (BrdU) dectection kit was purchased from Boehringer Mannheim.

Cell Culture
Rat aortic smooth muscle cells from thoracic aortas of 2- to 3-month-old Sprague-Dawley rats were prepared and cultured as described previously,¹⁸ and the procedures followed were in accordance with institutional guidelines. A monoclonal antibody against smooth muscle -actin was used to assess the purity of the smooth muscle cell cultures. Flow cytometry of anti–smooth muscle -actin antibody–stained cells revealed a purity of 95±3%. For all experiments early passaged (5 or less) VSMCs were used, and each individual experiment represented in the n value was performed with an independent preparation of VSMC.

Migration
VSMC migration was examined in transwell cell culture chambers with gelatin-coated polycarbonate membranes as described previously.¹⁸ In this assay, movement of VSMC through the coated membrane toward chemoattractants measures both invasion and chemotaxis. Cells were pretreated with PD98059 (10 or 30 µmol/L), TRO (1 to 20 µmol/L), RSG (0.1 to 10 µmol/L), or vehicle (0.4% FBS/DMEM) for 30 minutes at 37°C. Cell attachment to the gelatin-coated membrane was not affected by any of the inhibitors (data not shown). Inhibitors were added to both the upper and the lower compartments and were present throughout the duration of the experiment. Migration was induced by addition of TNF- (50 to 400 U/mL) to the lower compartment. After a 4-hour migration period, cells that had undergone migration toward the lower membrane surface were counted per x320 high power field (HPF) with the use of an Axiovert 135 microscope. Four randomly chosen HPFs were counted per membrane. Experiments were performed in duplicate or triplicate and were repeated at least 3 times.

Western Blot Analysis
For analysis of phosphorylated and activated MAPK, cultured VSMC were grown to 60% to 70% confluence and then starved for 24 hours in 0.4% FBS/DMEM. For inhibitor studies, cells were pretreated for 30 minutes with PD98059 (1 to 30 µmol/L), TNF- receptor-1 antibody (1:100–1:1000), TNF- receptor-2 antibody (1:100–1:1000), TRO (20 µmol/L), RSG (10 µmol/L), or vehicle (0.4% FBS/DMEM) alone, followed by the addition of TNF- (100 U/mL). After protein extraction, equal amounts of protein (30 µg) were separated by SDS-PAGE (7.5% standard gel), and Western blot analysis was performed as described previously¹⁸ with rabbit polyclonal antibodies that recognize either (a) ERK1 or ERK2, which are phosphorylated on threonine 202 and tyrosine 204, that is, phospho ERK/ERK2 MAPK, or (b) all ERK1 and ERK2 proteins, independent of their phosphorylation state, that is, total ERK1/ERK2 MAPK, or goat polyclonal antibodies against TNF- receptor-1 or TNF- receptor-2 protein. Antibodies were used at a concentration of 1:1000. To control for equal protein concentrations in MAPK experiments, 2 gels for each group were loaded in parallel with the same protein samples and blotted for activated, phosphorylated ERK1/ERK2 MAPK or total ERK1/ERK2 MAPK. All Western blot experiments were repeated at least twice with a different cell preparation.

BrdU Incorporation
To determine the effect of TNF- on DNA-synthesis, incorporation of the thymidine analogue BrdU was measured¹⁹ with the BrdU labeling and detection kit II from Boehringer Mannheim. VSMC were incubated either with TNF- (100 U/mL) in serum-free medium or were kept in serum-free medium as control. Cell nuclei incorporating BrdU appeared brown and were counted in 4 to 6 different HPFs per well and related to total cell number.

Statistics
ANOVA or paired or unpaired t test was performed for statistical analysis as appropriate. A probability value <0.05 was considered to be statistically significant. Data are expressed as mean±SEM.

	Results

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TNF- Stimulates VSMC Migration
To investigate the directed migration responses of VSMC toward TNF-, experiments were performed with a transwell migration chamber assay. Using increasing concentrations of TNF- (50 to 400 U/mL), we observed a dose-dependent increase in directed migration of cultured VSMC (Figure 1). The cytokine induced a modest response at 50 U/mL with 7.2±1.2 cells/HPF, representing a 1.7±0.3-fold stimulation compared with control (4.2±0.3 cells/HPF). Significantly augmented migration occurred at 100 U/mL and 200 U/mL TNF-, leading to a 2.74±0.13-fold and 3.4±0.1-fold stimulation, respectively (both P<0.05 compared with control). At the highest concentration tested, there was no further increase in migration toward TNF- (400 U/mL) (3.38±0.14-fold over control, P<0.05).

View larger version (44K): [in this window] [in a new window]   Figure 1. VSMC migration toward increasing concentrations of TNF- (50 to 400 U/mL). Migration of VSMC was determined with the use of a modified Boyden chamber assay as described in "Methods" and is shown in cell count per HPF. Results represent at least 3 independent experiments done in duplicate or triplicate. Data are expressed as mean±SEM. *P<0.05 vs control. Co indicates control.

TNF-–Induced Migration Is MAPK-Dependent
Although an induction of the ERK1/ERK2 MAPK pathway by TNF- in VSMC had not yet been demonstrated, MAPK activation is known to be an important step in PDGF-directed and angiotensin II–directed migration.^{18 20} In addition, TNF- has been shown to induce MAPK activation in several other cell types including fibroblasts,^{21 22} for which the cytokine is chemotactic.⁵ We therefore tested the specific MEK inhibitor PD98059 to examine a possible involvement of the MEK/MAPK pathway in TNF-–induced migration. The data in Figure 2 demonstrate that treatment of VSMC with the MAPK pathway inhibitor PD98059 at 10 µmol/L and 30 µmol/L significantly inhibited their response toward TNF- (100 U/mL) (66±7.2% and 100±6%, respectively; P<0.05). At the concentrations used, PD98059 did not cause any cytotoxic effects. There was no evidence of cell detachment or loss of plasma membrane integrity, as evidenced by the uptake of trypan blue.

View larger version (36K): [in this window] [in a new window]   Figure 2. Migration of VSMC toward TNF- is MAPK dependent. Cells were incubated with the MEK inhibitor PD98059 for 30 minutes before TNF- (100 U/mL) was added. MAPK inhibition with PD98059 at 10 µmol/L significantly reduced migratory response and abolished TNF-–induced migration at 30 µmol/L. VSMC migration is shown as x-fold induction over control. Experiments were repeated 3 times and done in duplicate. Data are expressed as mean±SEM. *P<0.05 vs TNF- alone.

TNF- Transiently Activates MAPK
To corroborate our findings that the pharmacological inhibitor PD98059 of the MAPK pathway inhibited TNF-–induced chemotactic signaling in VSMC, we examined the effect of TNF- on MAPK activation. VSMC were made quiescent by serum starvation, and after stimulation with TNF- the activation and phosphorylation of MAPK was assessed by immunoblotting with a phosphospecific ERK1/ERK2 MAPK antibody. In parallel experiments, the amount of total ERK1/ERK2 MAPK was determined in the same cell extracts with the use of an antibody that recognizes all ERK1/ERK2 MAPKs independent of their phosphorylation state. Quiescent cells in the control groups exhibited low MAPK activity, as evidenced by the faint bands detected with the phosphospecific antibody (Figure 3). The residual MAPK activity in the controls probably is due to the fact that a small percentage of serum (0.4%) was present, which is known to stimulate MAPK. Stimulation with TNF- for 10 minutes induced MAPK activation in a dose-dependent manner, resulting in a 11.5±2.3-fold increase in phosphorylated, activated MAPK compared with untreated control at a concentration of 100 U/mL (P<0.05). At higher concentrations of TNF-, no further increase in MAPK activation was observed, indicating that TNF-–inducible MAPK activity had plateaued (Figure 3). Interestingly, TNF- at 200 U/mL induced more VSMC migration than was observed in cells stimulated with 100 U/mL TNF-. Increased VSMC migration at concentrations of TNF- higher than required to induce maximal MAPK activation therefore may be mediated through pathways other than MAPK.

View larger version (54K): [in this window] [in a new window]   Figure 3. TNF- stimulates MAPK activation in a dose-dependent manner. Quiescent VSMC were stimulated with increasing concentrations of TNF- (10 to 400 U/mL) for 10 minutes. Equal amounts of protein (30 µg) were separated on 7.5% PAGE-SDS gels, followed by immunoblotting for (I) activated, phosphorylated ERK1/ERK2 MAPK or (II) total ERK1/ERK2 MAPK (top, representative immunoblots). Results of densitometric analysis of 3 different experiments are shown in the bottom panel. MAPK activity was measured by densitometry and is expressed in arbitrary units. Data are shown as mean±SEM. *P<0.05 vs control. Co indicates control.

Since different growth factors and peptides activate MAPK either transiently (5 to 10 minutes peak)¹⁹ or induce an additional second sustained peak in MAPK activity (5 to 10 minutes initial peak, followed by second peak 1 to 2 hours later),^{23 24} we performed a time-course study of MAPK induction by TNF-. As shown in Figure 4, TNF- (100 U/mL) induced a rapid and transient activation of MAPK that returns to baseline values within 60 minutes. No second peak or sustained late phase in TNF-–stimulated MAPK activity was detected. TNF- did not affect the amount of total MAPK protein during the investigated time course (Figure 4 lane II).

View larger version (37K): [in this window] [in a new window]   Figure 4. MAPK activation by TNF- is monophasic and transient. Serum-starved VSMC were treated with TNF- (100 U/mL) for 10 to 180 minutes, and protein samples were immunoblotted with I, a phosphospecific ERK1/ERK2 MAPK antibody, or II, an antibody against total ERK1/ERK2 MAPK. Western blots shown are representative of 4 experiments with different cell preparations. Co indicates control.

Treatment with PD98059 (1 to 30 µmol/L) blocked TNF-–stimulated MAPK activation in a concentration-dependent manner (Figure 5). TNF-–induced MAPK activation was inhibited by 37±5% and 74±8% at 1 µmol/L and 10 µmol/L PD98059, respectively. At 30 µmol/L, PD98059 completely abolished the effect of TNF- on MAPK activation (P<0.05).

View larger version (48K): [in this window] [in a new window]   Figure 5. PD98059 dose-dependently inhibits TNF-–induced MAPK activation. Cells were incubated with MEK-inhibitor PD98059 (1 to 30 µmol/L) for 30 minutes before TNF- (100 U/mL) was added. After stimulation with TNF- for 10 minutes, cell lysates were immunoblotted for I, activated, phosphorylated ERK1/ERK2 MAPK, or II, total ERK1/ERK2 MAPK (top, representative immunoblots). Results of densitometric analysis of 3 different experiments are shown in the bottom panel. MAPK activity was measured by densitometry and is expressed in arbitrary units. Data are shown as mean±SEM. *P<0.05 vs TNF- alone. Co indicates control.

TNF- Stimulates MAPK in VSMC Through p55 TNF- Receptor-1
To identify the receptor that mediates TNF-–induced MAPK activation, quiescent cells were pretreated with different concentrations of antibodies against the p55 TNF- receptor-1 and the p75 TNF- receptor-2 and then stimulated with TNF- (100 U/mL, 10 minutes). Whereas TNF-–stimulated MAPK activity was inhibited by increasing concentrations of the TNF- receptor-1 antibody (38±7% inhibition at 1:500 and 68±11% inhibition at 1:100), the TNF- receptor-2 antibody had no effect (Figure 6). An additional Western blot analysis of the same control extracts confirmed the presence of both receptors in VSMC (Figure 7). Thus, TNF- specifically activates MAPK in VSMC through the p55 TNF- receptor-1.

View larger version (45K): [in this window] [in a new window]   Figure 6. TNF- activates MAPK in VSMC through the p55 TNF- receptor-1. Quiescent rat aortic VSMC were pretreated for 30 minutes with increasing concentrations of p55 TNF- receptor-1 antibody (1:1000–1:100) or p75 TNF- receptor-2 antibody (1:1000–1:100) followed by addition of TNF- (100 U/mL). After coincubation with TNF- and receptor antibodies for 10 minutes, cells were harvested and protein samples were immunoblotted with I, a phosphospecific ERK1/ERK2 MAPK antibody, or II, an antibody against total ERK1/ERK2 MAPK. Western blots shown are representatives of 4 experiments with different cell preparations. Co indicates control.

View larger version (56K): [in this window] [in a new window]   Figure 7. Both TNF- receptors are present in VSMC. Control cell extracts were separated on 7.5% PAGE-SDS gels, followed by Western blot analysis with antibodies against p55 TNF- receptor-1 or p75 TNF- receptor-2. As shown, distinct bands of the right sizes were detected for both TNF- receptors. Experiment was repeated with a different set of protein samples, revealing the same results.

Thiazolidinediones Inhibit VSMC Migration Toward TNF-
Thiazolidinediones (TZDs) such as TRO are antidiabetic insulin sensitizers that have been reported to inhibit TNF- signaling in adipose tissue.²⁵ In addition, we have previously shown that TRO inhibits PDGF-mediated and angiotensin II–mediated migration of VSMC.^{19 20} Because of those findings, we examined the effects of the TZDs TRO and rosiglitazone RSG on TNF-–induced migration. The data in Figure 8 show that migration of VSMC toward the cytokine was significantly inhibited by treatment with TRO 10 µmol/L and 20 µmol/L by 68±5.8% and 99±7.2%, respectively (both P<0.05 vs TNF- 100 U/mL alone). An even more potent effect was observed for RSG that completely abolished migration toward TNF- at 10 µmol/L (P<0.05) (Figure 8).

View larger version (56K): [in this window] [in a new window]   Figure 8. Migration of VSMC toward TNF- is inhibited by TZDs troglitazone (TRO, 1 to 20 µmol/L) and rosiglitazone (RSG, 0.1 to 10 µmol/L) in a concentration-dependent manner. Migration responses are shown as x-fold induction over control. Results represent 4 independent experiments done in duplicate. Data are expressed as mean±SEM. *P<0.05 vs TNF- alone.

At all concentrations of TRO (1 to 20 µmol/L) or RSG (0.1 to 10 µmol/L) used in migration assays, we observed no cytotoxic effects evidenced by the lack of cell detachment or the absence of a significant number of cells staining positively for trypan blue.

TZDs Act Downstream of TNF-–Induced MAPK Activation
To determine whether TRO and RSG inhibited migration by targeting the MAPK pathway, we investigated their effect on TNF-–induced MAPK activation. VSMC were made quiescent by 24-hour serum starvation, then pretreated with TRO or RSG for 30 minutes, followed by stimulation with TNF- (100 U/mL) for 10 minutes. Neither TRO nor RSG attenuated the MAPK activation in response to TNF-, indicating that the TZDs inhibit VSMC migration downstream of MAPK activation, a signaling step required for TNF-–directed migration (Figure 9).

View larger version (45K): [in this window] [in a new window]   Figure 9. TZDs do not affect TNF-–induced MAPK activation. After a 30-minute preincubation with TRO (20 µmol/L) or RSG) 10 µmol/L), quiescent cells were stimulated with TNF- (100 U/mL) for 10 minutes. Immunoblotting was performed as described in Figure 3. I, Phosphospecific ERK1/ERK2 MAPK antibody; II, antibody against total ERK1/ERK2 MAPK. Western blots shown are representative of 2 independently performed experiments.

TNF- Is Not Mitogenic for VSMC
Because VSMC accumulation in restenosis and atherosclerosis results from a combination of cell growth and migration, it was of interest to determine whether TNF- also functions as a mitogen for VSMC. Using the thymidine analogue BrdU, we examined the effect of TNF- on VSMC proliferation. Treatment with TNF- (100 U/mL) for 24 hours did not result in a significant increase in DNA-synthesis compared with control, as determined by BrdU-incorporation (TNF-: 9.8±0.9%; control: 8.4±0.7%).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
TNF- is a cytokine that is released by inflammatory cells at sites of vascular injury and is expressed in the arterial wall under pathological conditions, in which it is associated with lesion formation.^{3 6 7 8 9 10 11} Migration of VSMC from the tunica media to the intima is one of the major pathological vascular responses that leads to the development and progression of intimal thickening.¹ Previous studies demonstrated that TNF- is a potent migration factor for different cell types, such as fibroblasts and inflammatory cells.^{4 5}

The present investigation demonstrates that (1) TNF- is a potent migration factor but not a growth factor for VSMC; (2) TNF- stimulates only an early activation (10 minutes) of MAPK but not a second, delayed peak in activity (2 to 6 hours), which may explain why this cytokine is a potent migration factor but a weak mitogen; (3) TNF-–induced MAPK activation is mediated by the p55 TNF- receptor and not the p75 TNF- receptor; and (4) TZDs inhibit TNF-–stimulated VSMC migration.

The mechanisms and intracellular signaling pathways leading to VSMC migration are not completely understood. Among the cytosolic events in response to migration factors, recent studies have recognized the ERK1/ERK2 MAPKs as key signaling steps for this process in VSMC.^{18 19 26} However, migration of VSMC has also been linked to other signaling molecules, such as increased phosphatidylinositol turnover leading to the activation of phospholipase C²⁷ as well as increased intracellular calcium and activation of the calcium/calmodulin-dependent kinase II.^{28 29 30 31} It has been shown that PDGF-induced chemotactic signaling involves activation of the calcium/calmodulin kinase II, which in turn leads to phosphorylation and activation of myosin light chain kinase.³² Yet, another recent study revealed that myosin light chain kinase, which phosphorylates and reorganizes cytoskeletal components that facilitate cell movement,³³ is also a substrate for MAPK.^{34 35} The importance of the MAPK-pathway in VSMC migration is supported by findings showing that inhibition of MAPK with antisense oligodeoxynucleotides or the MEK-inhibitor PD98059 blocks PDGF-directed and angiotensin II–directed migration.^{18 36}

TNF- is known to activate the MAPK pathway in fibroblasts²² and inflammatory cells²¹ ; however, nothing was known about its effects on MAPK in VSMC. Using the pharmacological inhibitor PD98059, we were able to identify that the activation of ERK1/ERK2 MAPK is a critical signaling step for TNF-–directed migration of VSMC. PD98059 selectively inhibits the dual-specificity kinase MEK, which phosphorylates and activates MAPK. In our experiments, PD98059, at the highest concentration tested, completely abolished both TNF-–induced MAPK activity and VSMC migration. The MEK inhibitor appears to be highly selective in blocking only the MAPK pathway; several studies have shown that it does not affect a number of other signaling proteins.^{37 38 39}

A variety of growth factors and peptides activate MAPK in different ways, leading either to a transient activation within 5 to 10 minutes after stimulation¹⁹ or to a biphasic response with a sustained second peak appearing 1 or 2 hours later.^{23 24} A recent study investigating the functional relevance of these kinetics reports that the early peak in PDGF-induced MAPK activation is crucial for VSMC migration, whereas the sustained second phase of MAPK activity is important for mitogenesis.²⁶ Potent chemoattractants such as PDGF¹⁸ and thrombin⁴⁰ that activate MAPK in a biphasic manner^{23 24} are also mitogenic for VSMC.^{41 42} Our data show that TNF- induces only the early peak of MAPK activity, leading to directed migration. No second peak of MAPK activity was observed, and consistent with other studies,⁴³ we saw no effect of TNF- to stimulate VSMC proliferation. However, some growth factors, such as FGF, which rapidly activate MAPK, do not induce migration of VSMC.¹⁹ This may indicate that MAPK activation is necessary but not sufficient for migration.

To further elucidate the signaling steps involved with TNF-–induced MAPK activation, we examined the role of the TNF- receptors. In other cell types, 2 main TNF- receptors have been described: the p55 TNF- receptor-1 (TNF--R1), and the p75 TNF- receptor-2 (TNF--R2).⁴⁴ Although many cell types express both receptors,⁴⁵ the majority of TNF-–induced signaling events are mediated by TNF--R1.⁴⁶ Using antibodies to neutralize the TNF--R1 and the TNF--R2, we found that only the TNF--R1 antibody blocked MAPK activation by TNF-, whereas the TNF--R2 had no effect. This finding is consistent with another study that showed that TNF--R1 mediated MAPK activation in HeLa cells.⁴⁵ In VSMC, TNF- induces signaling through the TNF--R1 that leads to MAPK activation, which is required for cell migration.

In addition to its role in directed migration toward TNF- and other chemoattractants, MAPK is also involved in mitogenic signaling by growth factors in VSMC. VSMC proliferation and migration both importantly contribute to the accumulation of these cells in vascular lesions. A potential candidate for a therapeutic approach for preventing lesion formation in the arterial wall could be the novel insulin-sensitizing class of TZDs. In the present study, we were able to demonstrate that 2 TZDs, TRO and RSG, inhibited TNF-–directed migration of VSMC. Furthermore, we previously showed that TRO inhibits PDGF-directed and angiotensin II–directed migration in VSMC^{19 20} and that migratory responses toward both chemoattractants are MAPK dependent.^{18 36} The precise mechanism by which TZDs inhibit MAPK-dependent migration pathways remains to be elucidated, although our results suggest an effect downstream of MAPK, since neither TRO nor RSG affected TNF-–stimulated MAPK activation.

Because of the enormous clinical relevance of VSMC accumulation in the development and progression of atherosclerosis and restenosis, strategies targeting the MAPK pathway as a common signaling step in VSMC migration and proliferation may provide new therapeutic approaches for the treatment and prevention of these vascular diseases.

	Acknowledgments

 
This study was supported by the Deutsche Forschungsgesellschaft (DFG GO 800/1-1) and the NIH (HL-58328-03). The authors would like to thank Verena Fromm for assistance in preparing the manuscript.

Received September 16, 1998; first decision October 14, 1998; accepted October 23, 1998.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Schwartz SM. Perspectives series: cell adhesion in vascular biology: smooth muscle migration in atherosclerosis and restenosis. J Clin Invest. 1997;99:2814–2816.[Medline] [Order article via Infotrieve]
2. Abedi H, Zachary I. Signalling mechanisms in the regulation of vascular cell migration. Cardiovasc Res. 1995;30:544–556.[Medline] [Order article via Infotrieve]
3. Warner SJ, Libby P. Human vascular smooth muscle cells: target for and source of tumor necrosis factor. J Immunol. 1989;142:100–109.[Abstract]
4. Furie MB, McHugh DD. Migration of neutrophils across endothelial monolayers is stimulated by treatment of the monolayers with interleukin-1 or tumor necrosis factor- alpha. J Immunol. 1989;143:3309–3317.[Abstract]
5. Postlethwaite AE, Seyer JM. Stimulation of fibroblast chemotaxis by human recombinant tumor necrosis factor alpha (TNF-alpha) and a synthetic TNF-alpha 31–68 peptide. J Exp Med. 1990;172:1749–1756.[Abstract/Free Full Text]
6. Raines EW, Ross R. Smooth muscle cells and the pathogenesis of the lesions of atherosclerosis. Br Heart J. 1993;69:S30–S37.
7. Jovinge S, Hultgardh-Nilsson A, Regnstrom J, Nilsson J. Tumor necrosis factor-alpha activates smooth muscle cell migration in culture and is expressed in the balloon-injured rat aorta. Arterioscler Thromb Vasc Biol. 1997;17:490–497.[Abstract/Free Full Text]
8. Clausell N, de Lima VC, Molossi S, Liu P, Turley E, Gotlieb AI, Adelman AG, Rabinovitch M. Expression of tumour necrosis factor alpha and accumulation of fibronectin in coronary artery restenotic lesions retrieved by atherectomy. Br Heart J. 1995;73:534–539.[Abstract/Free Full Text]
9. Barath P, Fishbein MC, Cao J, Berenson J, Helfant RH, Forrester JS. Tumor necrosis factor gene expression in human vascular intimal smooth muscle cells detected by in situ hybridization. Am J Pathol. 1990;137:503–509.[Abstract]
10. Rayment NB, Moss E, Faulkner L, Brickell PM, Davies MJ, Woolf N, Katz DR. Synthesis of TNF alpha and TGF beta mRNA in the different micro-environments within atheromatous plaques. Cardiovasc Res. 1996;32:1123–1130.[Abstract/Free Full Text]
11. Tanaka H, Swanson SJ, Sukhova G, Schoen FJ, Libby P. Smooth muscle cells of the coronary arterial tunica media express tumor necrosis factor-alpha and proliferate during acute rejection of rabbit cardiac allografts. Am J Pathol. 1995;147:617–626.[Abstract]
12. Clausell N, Molossi S, Sett S, Rabinovitch M. In vivo blockade of tumor necrosis factor-alpha in cholesterol-fed rabbits after cardiac transplant inhibits acute coronary artery neointimal formation. Circulation. 1994;89:2768–2779.[Abstract/Free Full Text]
13. Winkles JA, Gay CG. Regulated expression of PDGF A-chain mRNA in human saphenous vein smooth muscle cells. Biochem Biophys Res Commun. 1991;180:519–524.[Medline] [Order article via Infotrieve]
14. Yoshida S, Ono M, Shono T, Izumi H, Ishibashi T, Suzuki H, Kuwano M. Involvement of interleukin-8, vascular endothelial growth factor, and basic fibroblast growth factor in tumor necrosis factor alpha-dependent angiogenesis. Mol Cell Biol. 1997;17:4015–4023.[Abstract]
15. Couffinhal T, Duplaa C, Labat L, Lamaziere JM, Moreau C, Printseva O, Bonnet J. Tumor necrosis factor-alpha stimulates ICAM-1 expression in human vascular smooth muscle cells. Arterioscler Thromb. 1993;13:407–414.[Abstract/Free Full Text]
16. Loppnow H, Bil R, Hirt S, Schonbeck U, Herzberg M, Werdan K, Rietschel ET, Brandt E, Flad HD. Platelet-derived interleukin-1 induces cytokine production, but not proliferation of human vascular smooth muscle cells. Blood. 1998;91:134–141.[Abstract/Free Full Text]
17. Galis ZS, Muszynski M, Sukhova GK, Simon-Morrissey E, Libby P. Enhanced expression of vascular matrix metalloproteinases induced in vitro by cytokines and in regions of human atherosclerotic lesions. Ann N Y Acad Sci. 1995;748:501–507.[Abstract]
18. Graf K, Xi XP, Yang D, Fleck E, Hsueh WA, Law RE. Mitogen-activated protein kinase activation is involved in platelet- derived growth factor-directed migration by vascular smooth muscle cells. Hypertension. 1997;29:334–339.[Abstract/Free Full Text]
19. Law RE, Meehan WP, Xi XP, Graf K, Wuthrich DA, Coats W, Faxon D, Hsueh WA. Troglitazone inhibits vascular smooth muscle cell growth and intimal hyperplasia. J Clin Invest. 1996;98:1897–1905.[Medline] [Order article via Infotrieve]
20. Graf K, Xi XP, Hsueh WA, Law RE. Troglitazone inhibits angiotensin II-induced DNA synthesis and migration in vascular smooth muscle cells. FEBS Lett. 1997;400:119–121.[Medline] [Order article via Infotrieve]
21. Winston BW, Lange-Carter CA, Gardner AM, Johnson GL, Riches DW. Tumor necrosis factor alpha rapidly activates the mitogen-activated protein kinase (MAPK) cascade in a MAPK kinase kinase-dependent, c-Raf- 1-independent fashion in mouse macrophages. Proc Natl Acad Sci U S A. 1995;92:1614–1618.[Abstract/Free Full Text]
22. Vietor I, Schwenger P, Li W, Schlessinger J, Vilcek J. Tumor necrosis factor-induced activation and increased tyrosine phosphorylation of mitogen-activated protein (MAP) kinase in human fibroblasts. J Biol Chem. 1993;268:18994–18999.[Abstract/Free Full Text]
23. Hoshiya M, Awazu M. Trapidil inhibits platelet-derived growth factor-stimulated mitogen-activated protein kinase cascade. Hypertension. 1998;31:665–671.[Abstract/Free Full Text]
24. Rao GN, Delafontaine P, Runge MS. Thrombin stimulates phosphorylation of insulin-like growth factor-1 receptor, insulin receptor substrate-1, and phospholipase C-gamma 1 in rat aortic smooth muscle cells. J Biol Chem. 1995;270:27871–27875.[Abstract/Free Full Text]
25. Peraldi P, Xu M, Spiegelman BM. Thiazolidinediones block tumor necrosis factor-alpha-induced inhibition of insulin signaling. J Clin Invest. 1997;100:1863–1869.[Medline] [Order article via Infotrieve]
26. Nelson PR, Yamamura S, Mureebe L, Itoh H, Kent KC. Smooth muscle cell migration and proliferation are mediated by distinct phases of activation of the intracellular messenger mitogen-activated protein kinase. J Vasc Surg. 1998;27:117–125.[Medline] [Order article via Infotrieve]
27. Bornfeldt KE, Raines EW, Nakano T, Graves LM, Krebs EG, Ross R. Insulin-like growth factor-I and platelet-derived growth factor-BB induce directed migration of human arterial smooth muscle cells via signaling pathways that are distinct from those of proliferation. J Clin Invest. 1994;93:1266–1274.
28. Bilato C, Pauly RR, Melillo G, Monticone R, Gorelick-Feldman D, Gluzband YA, Sollott SJ, Ziman B, Lakatta EG, Crow MT. Intracellular signaling pathways required for rat vascular smooth muscle cell migration: interactions between basic fibroblast growth factor and platelet-derived growth factor. J Clin Invest. 1995;96:1905–1915.
29. Bilato C, Curto KA, Monticone RE, Pauly RR, White AJ, Crow MT. The inhibition of vascular smooth muscle cell migration by peptide and antibody antagonists of the alphavbeta3 integrin complex is reversed by activated calcium/calmodulin- dependent protein kinase II. J Clin Invest. 1997;100:693–704.[Medline] [Order article via Infotrieve]
30. Pauly RR, Passaniti A, Bilato C, Monticone R, Cheng L, Papadopoulos N, Gluzband YA, Smith L, Weinstein C, Lakatta EG, Crow MT. Migration of cultured vascular smooth muscle cells through a basement membrane barrier requires type IV collagenase activity and is inhibited by cellular differentiation. Circ Res. 1994;75:41–54.[Abstract/Free Full Text]
31. Pauly RR, Bilato C, Sollott SJ, Monticone R, Kelly PT, Lakatta EG, Crow MT. Role of calcium/calmodulin-dependent protein kinase II in the regulation of vascular smooth muscle cell migration. Circulation. 1995;91:1107–1115.[Abstract/Free Full Text]
32. Sobieszek A. Calmodulin-dependent autophosphorylation of smooth muscle myosin light chain kinase: intermolecular reaction mechanism via dimerization of the kinase and potentiation of the catalytic activity following activation. Biochemistry. 1995;34:11855–11863.[Medline] [Order article via Infotrieve]
33. Garcia JGN, Verin AD, Herenyiova M, English D. Adherent neutrophils activate endothelial myosin light chain kinase: role in transendothelial migration. J Appl Physiol. 1998;84:1817–1821.[Abstract/Free Full Text]
34. Klemke RL, Cai S, Giannini AL, Gallagher PJ, de Lanerolle P, Cheresh DA. Regulation of cell motility by mitogen-activated protein kinase. J Cell Biol. 1997;137:481–492.[Abstract/Free Full Text]
35. Morrison DL, Sanghera JS, Stewart J, Sutherland C, Walsh MP, Pelech SL. Phosphorylation and activation of smooth muscle myosin light chain kinase by MAP kinase and cyclin-dependent kinase-1. Biochem Cell Biol. 1996;74:549–557.[Medline] [Order article via Infotrieve]
36. Xi X-P, Graf K, Goetze S, Fleck E, Hsueh WA, Law RE. Central role of the MAP kinase pathway in AII-mediated DNA synthesis and migration in rat vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 1999. In press.
37. Dudley DT, Pang L, Decker SJ, Bridges AJ, Saltiel AR. A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc Natl Acad Sci U S A. 1995;92:7686–7689.[Abstract/Free Full Text]
38. Pang L, Sawada T, Decker SJ, Saltiel AR. Inhibition of MAP kinase kinase blocks the differentiation of PC-12 cells induced by nerve growth factor. J Biol Chem. 1995;270:13585–13588.[Abstract/Free Full Text]
39. Lazar DF, Wiese RJ, Brady MJ, Mastick CC, Waters SB, Yamauchi K, Pessin JE, Cuatrecasas P, Saltiel AR. Mitogen-activated protein kinase kinase inhibition does not block the stimulation of glucose utilization by insulin. J Biol Chem. 1995;270:20801–20807.[Abstract/Free Full Text]
40. Noda-Heiny H, Sobel BE. Vascular smooth muscle cell migration mediated by thrombin and urokinase receptor. Am J Physiol. 1995;268:C1195–C1201.[Abstract/Free Full Text]
41. Molloy CJ, Pawlowski JE, Taylor DS, Turner CE, Weber H, Peluso M. Thrombin receptor activation elicits rapid protein tyrosine phosphorylation and stimulation of the raf-1/MAP kinase pathway preceding delayed mitogenesis in cultured rat aortic smooth muscle cells: evidence for an obligate autocrine mechanism promoting cell proliferation induced by G-protein-coupled receptor agonist. J Clin Invest. 1996;97:1173–1183.[Medline] [Order article via Infotrieve]
42. Robinson CJ, Scott PH, Allan AB, Jess T, Gould GW, Plevin R. Treatment of vascular smooth muscle cells with antisense phosphorothioate oligodeoxynucleotides directed against p42 and p44 mitogen-activated protein kinases abolishes DNA synthesis in response to platelet-derived growth factor. Biochem J. 1996;320:123–127.
43. Stein CS, Fabry Z, Murphy S, Hart MN. Involvement of nitric oxide in IFN-gamma-mediated reduction of microvessel smooth muscle cell proliferation. Mol Immunol. 1995;32:965–973.[Medline] [Order article via Infotrieve]
44. Barbara JA, Van ostade X, Lopez A. Tumour necrosis factor-alpha (TNF-alpha): the good, the bad and potentially very effective. Immunol Cell Biol. 1996;74:434–443.[Medline] [Order article via Infotrieve]
45. Natoli G, Costanzo A, Moretti F, Fulco M, Balsano C, Levrero M. Tumor necrosis factor (TNF) receptor 1 signaling downstream of TNF receptor-associated factor 2: nuclear factor kappaB (NFkappaB)-inducing kinase requirement for activation of activating protein 1 and NFkappaB but not of c-Jun N-terminal kinase/stress-activated protein kinase. J Biol Chem. 1997;272:26079–26082.[Abstract/Free Full Text]
46. Wiegmann K, Schutze S, Kampen E, Himmler A, Machleidt T, Kronke M. Human 55-kDa receptor for tumor necrosis factor coupled to signal transduction cascades. J Biol Chem. 1992;267:17997–18001.[Abstract/Free Full Text]

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